The challenging disease management of tuberculosis (TB), a bacterial disease caused by Mycobacterium tuberculosis, and the increasing abundance of multi/extensive drug-resistant strains has precipitated to a severe public health problem. While global this mostly affects developing countries and regions with prevalent HIV/AIDS problems, and leads to an urgent need to find novel anti-TB lead compounds. The lack of success in finding such compounds by screening synthetic libraries directs the focus of interest back to the chemically diverse natural products. The constraints of classical bioactivity guided isolation are labor intensity, time commitment, and most important, the difficulty in detecting, isolating, and characterizing bioactive minor constituents. The present exploratory project will lay the groundwork for a transformative concept to the rapid detection, isolation, and identification of anti-tuberculosis natural products. The technology will be developed on extracts of cultured actinomycete strains and will be a prerequisite for a large-scale anti- TB drug discovery project on a panel of pre-selected anti-TB active actinomycete strains. A novel TLC-MS-(TB)-bioautography method will be developed for the straight-forward identification of active key compounds in an analytical setting that allows the mass spectrometric analysis of planar chromatographic sample separations and in parallel the evaluation of their biological activity profile. Combining the mass spectroscopic with biological activity data will generate two-dimensional maps that directly correlate the anti-TB activity with structural information. The isolation procedure can significantly be shortened by using the knowledge of the chemical properties of the active principle in an early stage of the separation procedure. A specific protocol can be tailored with counter current chromatography based on the polarity based GUESS principle and/or to directly upscale to preparative TLC. State-of-the-art technology in planar chromatography (high-performance TLC, automated spray-on sampling, gradient elution, semi-automatic extraction), microbiology (TB agar-overlay with luminescence detection), and MS spectroscopy (LC-MS2) will be used to target the isolation of lead compounds. Known chemical structures will be dereplicated based on their molecular weight, as gleaned from MS (and LC-MS2) and EI database (NIST, Wiley) search. Full structure elucidation of new or otherwise important isolated active principles will utilize state-of-the-art 1D/2D NMR- and MS-based structural information and will be supported by novel methodology like cryoprobe NMR, nuclear fingerprinting and purity-activity relationships based on quantitative NMR and modern anti-TB activity assessment. TLC-MS-(TB)-bioautography will be compared with established technologies, optimized, and validated. The concept can generally be applied in natural product drug discovery to handle large quantities of samples and will in our case be applied to pre-selected anti-TB active actinomycete strains. We expect that application of this novel technology will result in the discovery of a plethora of new, heretofore unidentified anti-TB natural products and that one or more of these will be a viable lead compound for TB drug development.